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Beta-cyclodextrin/polyamide-amine arborescence macromolecule/gold nano particle compound and preparation and application thereof

A technology of amine dendrimers and gold nanoparticles, which is applied in the field of nanocomposites and its preparation and application, to achieve the effects of reducing cytotoxicity, good biocompatibility, and improving biocompatibility

Inactive Publication Date: 2016-06-15
DONGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Retrieval of relevant literature and patents at home and abroad shows that the method of using β-CD modified dendrimers and their gold nanoparticles as carriers to load siRNA for gene transfection of malignant glioma cells (U87MG) has not been reported yet.

Method used

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  • Beta-cyclodextrin/polyamide-amine arborescence macromolecule/gold nano particle compound and preparation and application thereof
  • Beta-cyclodextrin/polyamide-amine arborescence macromolecule/gold nano particle compound and preparation and application thereof
  • Beta-cyclodextrin/polyamide-amine arborescence macromolecule/gold nano particle compound and preparation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Weigh 90.3mg of CDI and 63.2mg of β-CD and dissolve them in 5mL of DMSO, respectively, and stir the reaction for 6h. Then, 5 mL of DMSO solution in which 57.9 mg of the fifth-generation polyamidoamine dendrimer (G5PAMAM) was dissolved was added dropwise to the solution, and stirred at room temperature for 3 days. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cut-off of 8000-14000Da, dialyzed in phosphate (PBS) buffer solution for 1 day, then replaced with ultrapure water for dialysis for 2 days, and finally freeze-dried to obtain a white Powder product G5.NH 2 -β-CD. Then weigh 39.5mg of G5.NH 2 - β-CD was dissolved in 5 mL of ultrapure water, and 322.7 μL of 30 mg / mL chloroauric acid aqueous solution (HAuCl 4 4H 2 O), after continuing to stir for 30min, quickly add 445μL of 10mg / mL sodium borohydride (NaBH 4 ) ice solution to continue the reaction for 4h. Finally, the reaction solution was transferred to a dia...

Embodiment 2

[0044] G5PAMAM, AuDENPs prepared in Comparative Example 1 and AuDENPs-β-CD prepared by the method of Example 1 (marked as Q0, Q1 and Q2 respectively) were 0.25, 0.5, 1, 2, 3 according to the N / P ratio , 4 and 5, the siRNA was 1 μg / well to prepare complexes for gel retardation experiments. Prepare 1% eight-well agarose gel containing ethidium bromide (EB), add 5 μL of Marker control group to the first well of the agarose gel, and add the above-mentioned complex solution to the remaining 2-8 wells in sequence. Electrophoresis was carried out at 80V for 45min, and then photographed and analyzed at 312nm by an ultraviolet gel analysis system. The results showed that all carriers showed the ability to completely block siRNA when the N / P ratio was more than 2:1, and each carrier was resistant to Bcl-2siRNA (attached figure 1 ) is slightly weaker than that of VEGFsiRNA (attached figure 2 ), and under the same N / P ratio, Q2 has a stronger ability to compress and wrap siRNA than Q0...

Embodiment 3

[0046] G5PAMAM, AuDENPs prepared in Comparative Example 1 and AuDENPs-β-CD prepared by the method of Example 1 (marked as Q0, Q1 and Q2 respectively) were prepared under different N / P ratio conditions (2.5:1, 5: 1 and 10:1) respectively formed carrier / siRNA complexes with 5 μg siRNA through electrostatic interaction, incubated at room temperature for 15-30 min, and then added 1 mL of LPBS buffer. The hydrodynamic particle size and surface potential were characterized by a Malvern laser particle size analyzer (Malvern, MK, 633nm laser). The results showed that under the conditions of three experimental N / P ratios, the carrier was effective for Bcl-2siRNA (attached image 3 a) and VEGFsiRNA (attached image 3 b) The hydrodynamic particle size of the composites decreases with the increase of N / P ratio. And the carrier is against Bcl-2siRNA (attached Figure 4 a) and VEGFsiRNA (attached Figure 4 b) The overall change trend of the surface potential of the complexes is roughly ...

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Abstract

The invention relates to a beta-cyclodextrin / polyamide-amine arborescence macromolecule / gold nano particle compound and preparation and application thereof. The compound is formed by covalent binding between beta-cyclodextrin beta-CD and the fifth generation polyamide-amine arborescence macromolecule surface amino groups and loading gold nano particles. The water solution of the prepared G5. NH2-beta-CD is added into a chloroauric acid water solution dropwise for reaction, then an icy water solution of sodium borohydride NaBH4 is added for continuous reaction, and dialysis, freezing and drying are performed to obtain the beta-cyclodextrin / polyamide-amine arborescence macromolecule / gold nano particle compound. The compound AuDENPs-beta-CD serves as a nonviral vector to load siRNA to perform application of gene transfection. The transfection process is easy to operate, good in biocompatibility and high in transfection efficiency, and the compound has wide application potential on the aspect of biomedicine.

Description

technical field [0001] The invention belongs to the field of nanocomposites and their preparation and application, in particular to a β-cyclodextrin / polyamide-amine dendrimer / gold nanoparticle composite and its preparation and application. Background technique [0002] RNA interference (RNAi) refers to the phenomenon of gene silencing after specific gene transcription caused by double-stranded RNA (dsRNA) homologous to the target gene sequence. In cells, dsRNA-specific nucleases cleave it into small interfering RNA (siRNA) of no more than 21-25 nucleotides, and then mediate the degradation of specific mRNA, resulting in the blocking of corresponding gene expression and causing Gene silencing. This technique has been widely used in gene therapy research. [0003] Gene therapy, as a new technology born from the combination of modern medicine and molecular biology, is to correct or compensate diseases caused by gene deletion or abnormality by introducing exogenous normal gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08L87/00C08K3/08C08G81/00C08G83/00C08B37/16C12N15/87
CPCC08K3/08C08B37/0012C08G81/00C08G83/004C08K2003/0831C08K2201/011C12N15/87C08L87/00
Inventor 史向阳邱洁茹孔令丹李爱军曹雪雁
Owner DONGHUA UNIV
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